Salidroside Determination by HPLC using Uv-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 
 of salidroside in raw materials and finished product dietary supplements using HPLC and
 
 UV/VIS spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of salidroside in raw materials and
 
 finished products. Salidroside is a good chromophore and was measured at 205, other
 wavelengths should not be used without justification. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC Chemists to follow this procedure. 
 
 3.2 It is the responsibility of the QC Laboratory Management to ensure that this procedure is
 being followed. 
 
 3.3. It is the responsibility of the QC Laboratory and Analytical Development Management
 
 to keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 HPLC — High Performance Liquid Chromatography 
 
 4.2 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.3. HsPQs4-—- Phosphoric Acid 
 
 4.4 KH2PO«4 — Potassium phosphate monobasic 
 
 4.5 © ACN-— Acetonitrile 
 
 4.6 CofA — Certificate of Analysis 
 
 4.7. H20- Millipore Water 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev Page 
 Salidroside Determination by HPLC using UV/VIS 
 D-758 1 2 of 6 
 Spectroscopy 
 4.8 Salidroside — 2-(4-hydroxypheny])ethyl B-D-glucopyranoside 
 
 5.0 References 
 
 5.1 | USP43-NF38 USP Monograph for Rhodiola rosea 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 ore H20 
 
 oe ee ACN 
 
 6.1.3 H3PO4 
 
 6.1.4 KH2PO4 
 
 6.1.5 Methanol 
 
 6.1.6 Salidroside reference standard 
 
 6.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 622 Scintillation Vials 
 
 6.2.3 1L Mobile Phase Container 
 
 6.2.4 50mL Volumetric Flask 
 
 6.2.5 100mL Volumetric Flask 
 
 6.3. Disposables 
 
 6.3.1 10mL Pipette Tips 
 
 6.3.2 ImL Pipette Tips 
 
 6:3:3 200uL Pipette Tips 
 
 6.3.4 1.5mL microfuge tubes 
 
 6.3.5 16mL Test Tubes 
 
 6.3.6 Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev Page 
 Salidroside Determination by HPLC using UV/VIS 
 D-758 1 3 of 6 
 Spectroscopy 
 6.3.7 Nylon Syringe Filters, 0.2um 
 
 6.3.8 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical Balance 
 
 6.4.3 Ultrasonic bath 
 
 6.4.4 Vortex 
 
 6.4.5 Stir Plate 
 
 6.4.6 Eppendorf Centrifuge 
 
 6.4.7 10mL Pipette 
 
 6.4.8 ImL Pipette 
 
 6.4.9 200uL Pipette 
 
 7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards
 
 Tal Mobile Phase A — H2O0 
 
 G2 Mobile Phase B —- ACN 
 
 7.3 Diluent—100% methanol 
 
 7.4 Working Standard Preparation 
 
 7.4.1 Accurately weigh and transfer about 30 mg of Salidroside reference standard into
 
 a 100-mL volumetric flask. 
 
 7.4.2 Dissolve in and QS with Diluent. 
 
 is Sample Preparation 
 
 7.5.1 Raw materials can be dissolved in Diluent at any volume starting from 50 mL and
 
 any weight greater than the minimum weight of the analytical balance.
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev Page 
 Salidroside Determination by HPLC using UV/VIS 
 D-758 1 4 of 6 
 Spectroscopy 
 7.5.2 Accurately weigh and transfer an amount of sample sufficient to generate a
 salidroside concentration of 0.3 mg/mL into a suitably sized volumetric flask.
 
 7.5.3 Dilute to volume using Diluent, and sonicate for 10 minutes.
 
 7.5.4 Before injection, insoluble matter should be removed via filtration using a 0.45um
 
 nylon syringe filter. Discard at least 0.5 mL of the filtrate before collecting a
 
 portion in an HPLC vial for analysis. 
 
 7.5.4.1 Alternatively, samples and standards can also be centrifuged at 5000
 RPM for 3 minutes in an Eppendorf centrifuge to pellet insoluble matter.
 
 7.5.5 For raw materials or finished products being analyzed for the first time using this
 
 method, in-process verification is required to demonstrate spectral purity and
 
 extraction efficiency before the method can be implemented. 
 
 8.0 Test Conditions 
 
 8.1 Gradient 
 
 Time MA %B Gradient Type 
 
 0.00 94 6 Initial 
 
 6.00 83 17 Linear 
 
 7.00 80.3 19.7 Linear 
 
 9.00 80.3 197 0 
 
 10.0 0 100 Linear 
 
 12.0 94 6 Linear 
 
 17.0 94 6 0 
 
 8.2 Column — ZORBAX Eclipse AAA. 3.5 um, 120A, 4.6 X 250 mm, or equivalent
 
 8.3 Flow Rate — 1.0 mL/min 
 
 8.4 UV Detection — 205 nm 
 
 8.5 3D Spectral Range — 210 nm — 320 nm 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev Page 
 Salidroside Determination by HPLC using UV/VIS 
 D-758 5 of6 
 Spectroscopy 
 8.6 Injection Volume — 1 wL 
 
 8.7. Column Temperature — 40 °C 
 
 8.8 | Recommended Sequence 
 
 8.8.1 Make at least 2 injections of the diluent. 
 
 8.8.2 Make five (5) injections of Standard Solution. 
 
 8.8.3 Make a single injection of each Sample Preparation. 
 
 8.8.4 Make a single injection of the Standard Solution after every six (6) samples and
 
 at the end of the run. 
 
 8.9 System Suitability Requirements 
 
 8.9.1 The %RSD of five (5) injections of Working Standard is NMT 2%.
 
 8.9.2 The %RSD of all injections of Working Standard is NMT 3%. 
 
 8.9.3 The spectral match over the range 210 nm — 320 nm is NLT 900.
 
 8.9.4 The retention time of the sample is within 0.3 min of the standard.
 
 8.10 Column Wash and Storage 
 
 8.10.1 Rinse and store the column with H20/ACN (50/50). 
 
 9.0 Calculations 
 
 9.1 Example calculations for determining finished product % label or raw material % purity
 
 91.1 % assay = ~ x asWtetra d*XTP yg SV Ry LSS PPX 100 
 Vsta SA ° LA 
 
 Ry Sample peak area 
 
 Rg Mean standard peak area 
 
 Wtsq Weight of reference standard in mg 
 
 V;tq Volume of the standard preparation accounting for dilutions in
 
 mL 
 
 P Purity of the reference standard in decimal format
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev Page 
 Salidroside Determination by HPLC using UV/VIS 
 D-758 6 of6 
 Spectroscopy 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 Von. Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Weight of a single dosage unit in mg for tablets and
 
 capsules, volume of a single serving from the theoretical formula
 in mL for liquids, or 1 for raw materials. 
 
 LA Label amount in mg per dose or | for raw materials
 
 10.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New N/A J. Maignan Update for consistency with current practices and clarity. Fix standard concentration to match USP since linearity is not validated. 04/11/22 Add system suitability section. Add recommended sequence. Add CC-22-0175 S. Sassman column wash and storage. Add requirement for spectral match and set range. | - | - |